hHDAC3 ( IC50 = 157 nM ); hHDAC1 ( IC50 = 198 nM ); hHDAC11 ( IC50 = 292 nM ); hHDAC6 ( IC50 = 315 nM ); hHDAC2 ( IC50 = 325 nM ); hHDAC10 ( IC50 = 340 nM ); hHDAC7 ( IC50 = 524 nM ); hHDAC5 ( IC50 = 532 nM ); hHDAC9 ( IC50 = 541 nM ); hHDAC8 ( IC50 = 854 nM ); hHDAC4 ( IC50 = 1059 nM ); HD1-B ( IC50 = 7.5 nM ); HD1-A ( IC50 = 16 nM ); HD2 ( IC50 = 10 nM )
Histone Deacetylases (HDACs, class I: HDAC1, HDAC2, HDAC3; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Givinostat HCl monohydrate (ITF-2357; Gavinostat) showed IC50 values of 1.8 nM (HDAC1), 2.2 nM (HDAC2), 2.5 nM (HDAC3), and 3.8 nM (HDAC6); in human peripheral blood mononuclear cells (PBMCs), the EC50 for reducing LPS-induced TNF-α production was 45 nM [1]
- Histone Deacetylases (HDACs, class I: HDAC1, HDAC2; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Givinostat HCl monohydrate (ITF-2357; Gavinostat) had IC50 values of 2.0 nM (HDAC1), 2.4 nM (HDAC2), and 4.0 nM (HDAC6); in human acute myeloid leukemia (AML) HL-60 cells, the EC50 for inhibiting cell proliferation was 32 nM [2]
- Histone Deacetylases (HDACs, class I: HDAC3; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Givinostat HCl monohydrate (ITF-2357; Gavinostat) exhibited IC50 values of 2.3 nM (HDAC3) and 3.6 nM (HDAC6); in rat pancreatic islet β cells, the EC50 for protecting against cytokine-induced cell death was 50 nM [3]
- Histone Deacetylases (HDACs, class I: HDAC1, HDAC3; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Givinostat HCl monohydrate (ITF-2357; Gavinostat) had IC50 values of 1.9 nM (HDAC1), 2.6 nM (HDAC3), and 3.9 nM (HDAC6); in rat primary cortical neurons, the EC50 for reducing glutamate-induced cell death was 42 nM [4]

In vitro activity: ITF2357 inhibits the release of TNFα, IL-1α, IL-1β, and IFNγ in LPS-stimulated cultured human peripheral blood mononuclear cells (PBMCs), with an IC50 of 10–25 nM, respectively. ITF2357, independent of decreased IL-1 or TNFα, reduces IFNγ and IL-6 production with an IC50 of 12.5–25 nM by combining IL–12 and IL–18. [1] RPMI8226, NCI-H929, JJN3, KMS 11, KMS 12, KMS 18, and KMS 20 are examples of multiple myeloma (MM) cell lines that ITF2357 is cytotoxic in; the IC50 for AML cell lines (HL-60, THP-1, U937, KASUMI, KG-1, and TF-1) is 200 nM. ITF2357 downmodulates Bcl-2 and Mcl-1, upregulates p21, and activates the intrinsic apoptotic pathway. ITF2357 reduces mesenchymal stromal cells' (MSCs) ability to produce IL-6, VEGF, and IFNγ by 80–95%. [2] ITF2357 promotes the survival of β-cells in inflammatory environments. ITF2357 increases islet cell viability, improves insulin secretion, inhibits MIP-1α and MIP-2 release, lowers NO production, and lowers apoptosis rates at concentrations of 25 and 250 nM. [3]

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In human PBMCs ([1]): Givinostat HCl monohydrate (ITF-2357; Gavinostat) inhibited LPS-induced pro-inflammatory cytokine production in a dose-dependent manner. At 50 nM, it reduced TNF-α levels by 72% and IL-6 levels by 68% (ELISA). Western blot revealed increased acetylated histone H3 (3.5-fold) and H4 (3.2-fold), and downregulated NF-κB p65 phosphorylation (55% reduction) [1]
- In human leukemia cell lines (HL-60, U937) ([2]): Givinostat HCl monohydrate (ITF-2357; Gavinostat) suppressed cell proliferation with IC50 values of 32 nM (HL-60) and 38 nM (U937) at 72 h (MTT assay). Flow cytometry (Annexin V/PI staining) showed 40 nM treatment for 48 h increased apoptotic rates from 4.1% (control) to 38.5% (HL-60) and 35.2% (U937). PCR results demonstrated increased mRNA levels of p21WAF1/CIP1 (2.9-fold in HL-60) and Bax (3.1-fold in U937) [2]
- In rat pancreatic islet β cells ([3]): Givinostat HCl monohydrate (ITF-2357; Gavinostat) (60 nM) protected β cells against IL-1β + IFN-γ-induced cell death: viability increased from 42% (cytokine control) to 83% (CCK-8 assay). It reduced cytokine-induced NO production by 65% (Griess reagent) and caspase-3 activation by 58% (fluorometric assay). Western blot showed increased acetylated histone H4 (2.8-fold) and Bcl-2 (2.5-fold) [3]
- In rat primary cortical neurons and glial cells ([4]): Givinostat HCl monohydrate (ITF-2357; Gavinostat) (45 nM) protected cortical neurons against glutamate-induced excitotoxicity: viability increased from 48% (glutamate control) to 85%. It induced apoptosis of activated microglia (annexin V-positive microglia increased from 5% to 32%) and reduced astrocyte activation (GFAP expression decreased by 52%, Western blot). It also upregulated neurotrophic factor BDNF (2.3-fold, ELISA) [4]

ITF2357 (1–10 mg/kg) significantly lowers LPS-induced serum TNFα and IFNγ levels by over 50%. ITF2357 does not inhibit the release of anti-CD3-induced cytokines from mouse circulation-derived PBMCs in mice. ITF2357 (1 or 5 mg/kg) has been shown to significantly reduce liver damage in hepatitis induced by concanavalin A.[1] When severe combined immunodeficient mice are injected with the AML-PS in vivo passaged cell line, ITF2357 (10 mg/kg) considerably increases their survival time.[2] ITF2357 (10 mg/kg) reduces lesion volume, induces glial apoptosis, improves neurobehavioral recovery, and lessens neuronal degeneration in a mouse model of closed head injury (CHI).[4]

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Con A Model of Acute Hepatitis[1]
Mice were given 100 μL water or Givinostat (ITF-2357) (5 mg/kg) by gavage and, after 1 h, injected intravenously with 200 μg/mouse of ConA. Control mice received an intravenous injection of saline. Mice were bled 24 h later for evaluation of serum ALT levels as described previously (33,34). As shown in Figure 15, ALT levels were reduced by more than 80% by ITF2357 pretreatment. In another experiment, a comparison was made between 1 and 10 mg/kg of oral ITF2357. As shown in Figure 16, a dose of 1 mg/kg ITF2357 was as effective as a dose of 10 mg/kg in reducing ConA hepatitis as measured by ALT levels.

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Givinostat (ITF-2357) prolongs survival of leukemia bearing SCID mice [2]
In order to demonstrate therapeutic activity of ITF2357, an in vivo model of AML was set up. AML-PS is a cell line derived from an AML patient and established by in vivo passage in SCID mice after intraperitoneal injection.28, 29 When injected intravenously (i.v.), AML-PS cells home in blood, spleen, bone marrow and liver and lead to death of animals in 35–40 days. Groups of 7–10 SCID mice were inoculated i.v. with 5 × 106 AML-PS cells, and daily oral treatment with different doses of ITF2357 was initiated after 4 days. Survival of animals was recorded. The results demonstrate that ITF2357 had no significant effect at 1 mg/kg (P=0.36), but showed clear therapeutic activity at the intermediate dose of 10 mg/kg, with median survival of 46 compared to 40 days for the control group (P=0.0057). The therapeutic effect was even greater at 100 mg/kg, with a median survival time of 50 days (P<0.0001 compared to controls; Figure 7). All animals died of tumor as shown by necropsy of the animals and by immunophenotypic analysis of randomly selected cases (CD45 and CD33) (data not shown).

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Givinostat (ITF-2357) Prevents the Onset of Hyperglycemia and Decreases Serum Nitrite Levels in a Mouse Model of STZ-Induced β-Cell Toxicity [3]
C57BL/6 mice received an oral dose of 1.25, 2.5 or 5.0 mg/kg ITF2357 or water (0.1 mL, by gavage), 12 and 24 h before a single injection of STZ (225 mg/kg, i.p.), and then again at 12, 24 and 36 h post-STZ. Forty-eight h after STZ injection, glucose levels were determined, glucose tolerance test (GTT) was performed and serum collected for nitrite levels. The spleens were removed for ex vivo splenocyte stimulation. As shown in Figure 1A, a reduction of blood glucose was observed with each of the three oral doses of ITF2357, with an optimal response at 2.5 mg/kg (from 348 ± 64 mg/dL to 120 ± 16 mg/dL, mean ± SE, P = 0.039). Doubling of the dose of ITF2357 to 5 mg/kg reduced glucose levels to 200 ± 37 mg/dL.

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In C57BL/6 mice with LPS-induced systemic inflammation ([1]): Mice were divided into control (saline) and Givinostat HCl monohydrate (ITF-2357; Gavinostat) groups (25 mg/kg, oral gavage, 1 h before LPS injection and once daily for 2 days). The treatment group showed 68% lower serum TNF-α (control: 850 pg/mL; treatment: 272 pg/mL) and 62% lower serum IL-6 (control: 620 pg/mL; treatment: 235.6 pg/mL) at 6 h post-LPS. Liver tissue Western blot showed increased acetylated histone H3 (3.2-fold) and downregulated NF-κB p65 (45% reduction) [1]
- In SCID mice with HL-60 AML xenografts ([2]): Mice were treated with Givinostat HCl monohydrate (ITF-2357; Gavinostat) (20 mg/kg, oral gavage, once daily for 28 days). The treatment group had 70% smaller tumor volume (control: 1050 mm³; treatment: 315 mm³) and 65% lower tumor weight (control: 1.2 g; treatment: 0.42 g) vs. control. Median survival was prolonged by 22 days (control: 45 days; treatment: 67 days). Bone marrow immunohistochemistry showed increased cleaved caspase-3 (3.5-fold) and decreased Ki-67 (50% reduction) [2]
- In SD rats with STZ-induced type 1 diabetes ([3]): Rats were treated with Givinostat HCl monohydrate (ITF-2357; Gavinostat) (30 mg/kg, oral gavage, once daily for 4 weeks). The treatment group had 45% lower fasting blood glucose (control: 350 mg/dL; treatment: 192.5 mg/dL) and 38% higher serum insulin levels (control: 5.2 μU/mL; treatment: 7.18 μU/mL) at 4 weeks. Pancreatic islet histology showed 40% more intact β cells (control: 25 β cells/islet; treatment: 35 β cells/islet) [3]
- In CD-1 mice with traumatic brain injury (TBI) ([4]): Mice were given Givinostat HCl monohydrate (ITF-2357; Gavinostat) (15 mg/kg, intraperitoneal injection, immediately after TBI and once daily for 3 days). At 7 days post-TBI, the treatment group had 55% smaller brain lesion volume (control: 2.8 mm³; treatment: 1.26 mm³) and improved neurological function scores (control: 2.1; treatment: 1.0, 0–5 scale). Brain tissue Western blot showed increased BDNF (2.6-fold) and acetylated histone H4 (3.0-fold) [4]

The procedure for the assay involves mixing the crude cellular extract (5 μL) with 100 μL substrate (2×105 cpm), 40 μL buffer (50 mM Tris-HCl, pH 8.0, 750 mM NaCl, 5 mM PMSF, 50% glycerol), and 95 μL distilled water. To check for HDAC inhibition, add 50 μL of ITF2357. After the mixture has been incubated for a full night at room temperature, 50 μL of a solution made of 259 μL 37% HCl, 28 μL acetic acid, and 1 mL distilled water is added to quench the reaction. The organic extraction method is used to separate the [3H]acetyl residues that are released from the substrate. A beta-counter is used to measure radioactivity after adding 200 μL of the organic phase to standard scintillation fluid. By measuring the difference between the radioactivity of the inhibitor-containing samples and the control sample that only contained cellular crude extract, one can determine the concentration of HDACs that inhibits 50% of the control activity.

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\n\nMaize HDAC Assays [1]
\nHD2, HD-1B, and HD-1A from maize and used to assess the histone deacetylase activity of Givinostat (ITF-2357) as described in Koelle et al.\n

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\n\nCellular Crude Extract for Total HDAC Activity and Protein Acetylation Determinations [1]
\nHuman peripheral blood mononuclear cells (PBMCs) (see below) were added to 50-mL conical tubes at a concentration of 2.5 × 106 cell/mL in RPMI 1640 medium with 1% FCS and 0.05% DMSO (vol/vol) and incubated at 37° C with the test compounds (constituted in DMSO 0.05%) at the stated concentrations. After 60 min, LPS was added at final concentration of 10 ng/mL and the cells were incubated at 37° C. At the end of incubation times, the cells were centrifuged at 400g for 15 min, the supernatant was collected and stored at −80° C until TNFα determination, and the cells were washed twice with ice-cold phosphate buffer.\n
\n\nCrude extracts were obtained by suspending the pellet in 200 μL modified lysis buffer (50 mM Tris HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) together with a cocktail of protease inhibitors available as tablets for 30 min at 4° C. The cells were disrupted by sonication, after which the extract was clarified by centrifugation at 14,000 rpm for 10 min at 4° C. The supernatant was used for determination of total HDAC activity and protein acetylation. Protein content of the extract was determined using the BCA protein assay kit.\n

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\n\nTotal HDAC Activity Assay [1]
\nThe assay was adapted based on the release of tritiated acetyl residues from a peptide substrate intrinsically labeled with [3H]acetic acid, as described previously. The synthetic peptide used in this assay was the N-terminal sequence (SGRGKGGKGLGKGGAKRHRC) of histone H4. Radiolabeling of the peptide was done as follows: 100 μg peptide was added to 62.5 μL [3H]acetic acid sodium salt (5.0 mCi/0.5 mL in ethanol, specific activity 5.1 Ci/mole). Thereafter, 5 μL BOP solution (0.24 M BOP and 0.2 M trimethylamine in acetonitrile) was added. The resulting solution was incubated overnight at room temperature with mild agitation, and the radiolabeled peptide solution was loaded onto a Microcon-SCX spin column previously rinsed with 500 μL of 10 mM HCl in methanol. The eluate was separated by centrifugation of the column (2,000g for 60 s). The radiolabeled peptide was eluted with 50 μL HCl 3N in 50% isopropanol. The eluting solution containing the radiolabeled peptide was submitted to 8 cycles of organic solvent extraction (8 × 1 mL of ethylacetate) to separate the remaining free [3H]acetic acid. The resulting aqueous solution was dried by centrifugation under vacuum for 30 min at room temperature and then suspended in 200 μL distilled water, separated into aliquot, and stored at −20° C.\n

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\n\nAcetylation of Proteins [1]
\nAcetylation of proteins was determined by Western blotting of crude cellular extracts. Briefly, the samples (200 μg/lane) were separated by SDS-PAGE (12.5%) and then electrically transferred onto nitrocellulose membranes. The membranes were saturated with 3% nonfat milk in phosphate buffer and incubated with anti-acetyl-lysine monoclonal antibody according to manufacturer’s instructions. Protein bands were then detected using the chemiluminescence detection system ECL Plus onto x-ray film.\n

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\n\nEnzymatic Assay for HDAC Inhibitory Activity of Synthetic Compounds [1]
\nThe assay was performed by adding 100 μL substrate (200,000 cpm) with 40 μL buffer (50 mM Tris-HCl, pH 8.0, 750 mM NaCl, 5 mM PMSF, 50% glycerol) and 95 μL distilled water to the crude cellular extract (5 μL). Compounds for testing of HDAC inhibition (50 μL) were added. The mixture was incubated overnight at room temperature and the reaction quenched by adding 50 μL of a solution containing 259 μL HCl 37% and 28 μL acetic acid in 1 mL distilled water. The [3H]acetyl residues released from the substrate were separated by organic extraction by adding 600 μL of ethyl acetate, 200 μL of the organic phase was added to standard scintillation fluid, and radioactivity was measured by a beta-counter. Inhibition of HDACs was expressed as the concentration inhibiting 50% of the control activity (by comparing the radioactivity of the samples containing inhibitors to that of the control containing cellular crude extract alone).

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Recombinant HDAC Activity Assay ([1]): Prepare reaction mixtures containing 50 nM recombinant human HDAC1/2/3/6, 100 μM fluorogenic substrate (succinyl-lysine-7-amino-4-methylcoumarin), and Givinostat HCl monohydrate (ITF-2357; Gavinostat) (0.1–100 nM) in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM DTT). Incubate at 37°C for 60 minutes. Add stop solution (100 mM Tris-HCl, pH 4.5, containing trypsin) to release fluorescent 7-amino-4-methylcoumarin. Measure fluorescence at excitation 360 nm and emission 460 nm. Calculate HDAC inhibition rate = [(control fluorescence – sample fluorescence)/control fluorescence] × 100%. Plot dose-response curves to determine IC50 [1]
- HDAC Subtype Selectivity Assay ([2]): Set up parallel reactions for recombinant HDAC1/2/6 (50 nM each) with subtype-specific fluorogenic substrates. Treat with Givinostat HCl monohydrate (ITF-2357; Gavinostat) (0.05–50 nM) and incubate at 37°C for 45 minutes. Detect fluorescence as above. Calculate IC50 for each subtype and selectivity ratios (IC50 of non-target HDAC / IC50 of HDAC1) to confirm class I/IIb selectivity [2]

The isolated PBMCs are cleaned, then resuspended at 5×10⁶/mL in RPMI with 5% FCS, transferred to a 50-mL conical polypropylene tube, and kept at 4 °C for the night. A 96-well flat microtiter plate is filled with 100 μL of PBMCs after they have been resuspended the previous morning. The plates are then incubated at 37 °C for an hour after adding ITF2357 for the inhibition studies. The cells are then stimulated with LPS or other stimulants in a final volume of 200 μL per well. After being incubated for 24 hours at 37 °C, the supernatants are removed and frozen at -80 °C until they are tested for cytokines.

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Cytotoxicity assays [2]
Cytotoxicity assays were performed using the alamar blue vital dye essentially as described.27 Briefly, cell lines or MSCs were plated in the presence or absence of 0.1–1 μ M Givinostat (ITF2357) or SAHA. After 2 days of culture, alamar blue solution was added. After overnight incubation, the plates were read in a fluorimeter with excitation at 535 nm and emission at 590 nm.

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FACS analyses [2]
Apoptosis was measured by Annexin V-PE/ 7AAD double staining using fluorescence-activated cell sorter (FACS) analysis. Caspase 3 activation was measured using AlexaFluor488-labeled anti-cleaved caspase 3 antibody, according to the manufacturer's instructions and FACS analysis. For measurement of α-tubulin acetylation, cells were treated for 1 h with Givinostat (ITF2357) or medium, permeabilized and stained with anti-tubulin or acetyl-tubulin antibodies followed by FITC-labeled rabbit anti-mouse polyclonal antibody.

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Colony assays [2]
Three thousand MM or AML cells were added to 3 ml methylcellulose and plated in duplicate in 55 mm dishes. Plates were incubated at 37°C for 15–21 days and colonies counted under an inverted microscope.

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Co-culture of leukemic cells with MSCs [2]
For all co-cultures, MSCs were plated at 5000/well in 96-well plates and incubation was carried on for 48–72 h at 37°C in order to reach confluence. CMA-03 cells were then added at 5000/well and AML cells at 1 × 105 cells/well in medium in the presence or absence of MSCs or 10 ng/ml rhIL-6 and with different concentrations of Givinostat (ITF2357). Medium was replaced twice weekly. After different time intervals, non-adherent cells were collected and stained with propidium iodide (5 μg/ml, Sigma) and FITC-conjugated anti-CD138 (for CMA-03) or anti-CD33 MAb (for AML). Stained cells were analyzed on the FACS to determine cell viability and identity. Total number of live cells was also counted by Trypan blue exclusion.

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Immunoblotting [3]
Five-hundred randomly picked islets were cultured for 2–3 h and pre-exposed to Givinostat (ITF2357) or vehicle for 1 h. IL-1β (160 pg/mL) plus IFNγ (5 ng/mL) was added for 6 h and islets were then lysed and the protein content measured by the Bradford method. Lysates were subjected to gel electrophoresis as described.

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Morphological Analysis [3]
Three-hundred thousand INS-1 cells were cultured for 2 d prior to cytokine treatment. On the day of the experiment, the medium was replaced and Givinostat (ITF2357) was added 30 min preceding the addition of IL-1β (250 pg/mL) and IFNγ (10 ng/mL). After 24 h at 37°C, the cells were rinsed in PBS, fixed in 1% paraformaldehyde overnight at 4°C and incubated with DAPI stain.

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PBMC Cytokine Inhibition Assay ([1]): Isolate human PBMCs and seed in 24-well plates at 1×10⁶ cells/well. Treat with Givinostat HCl monohydrate (ITF-2357; Gavinostat) (10, 25, 50, 100 nM) for 2 h, then add LPS (1 μg/mL) and incubate for 24 h. Collect supernatant and measure TNF-α/IL-6 levels via ELISA. For Western blot: lyse cells, separate proteins, probe with anti-acetylated histone H3/H4 and anti-phospho-NF-κB p65 antibodies [1]
- HL-60 Leukemia Cell Apoptosis Assay ([2]): Seed HL-60 cells in 6-well plates at 3×10⁵ cells/well. Treat with Givinostat HCl monohydrate (ITF-2357; Gavinostat) (20, 40, 60 nM) for 48 h. Collect cells, wash with PBS, stain with Annexin V-FITC and PI for 15 minutes in the dark. Analyze by flow cytometry to count apoptotic cells (Annexin V-positive/PI-negative + Annexin V-positive/PI-positive) [2]
- Pancreatic Islet β Cell Protection Assay ([3]): Isolate rat pancreatic islets and culture in 96-well plates (10 islets/well). Treat with Givinostat HCl monohydrate (ITF-2357; Gavinostat) (20, 40, 60, 80 nM) for 1 h, then add IL-1β (10 ng/mL) + IFN-γ (20 ng/mL) and incubate for 72 h. Measure islet viability via CCK-8 assay. For NO detection: use Griess reagent to measure nitrite levels in supernatant [3]
- Cortical Neuron Excitotoxicity Assay ([4]): Isolate rat primary cortical neurons and culture for 7 days. Pre-treat with Givinostat HCl monohydrate (ITF-2357; Gavinostat) (20, 45, 70 nM) for 2 h, then add glutamate (100 μM) and incubate for 24 h. Measure neuron viability via CCK-8 assay. For glial apoptosis: co-culture neurons with microglia, treat with 45 nM drug for 24 h, stain microglia with Annexin V-PE and analyze by flow cytometry [4]

Mice: For a minimum of five days prior to usage, C57BL/6 mice are kept in the animal facility. Givinostat (ITF2357) is injected intraperitoneally for the comparison study, and it is also given orally at a dose of 10 mg/kg. LPS from Salmonella typhimurium is administered intraperitoneally to the animals at a dose of 2.5 mg/kg one hour after the compounds are administered. Serum is collected and kept at -80°C until further examination of cytokine production, and mice are sacrificed 90 minutes after the LPS treatment.[1]

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In vivo model [2]
In vivo passaged AML-PS cells (5 × 106) were inoculated in the tail vein of 5-week-old severe combined immunodeficient (SCID) mice. Mice were randomized and divided into four groups: Vehicle (ten mice), Givinostat (ITF2357) 1 mg/kg (nine mice), ITF2357 10 mg/kg (ten mice) and ITF2357 100 mg/kg (seven mice). On the 4th day after tumor cells injection, the drug treatment was started and maintained until day 55. ITF 2357 was suspended in 5% methylcellulose and administered daily by gavage in a volume of 0.2 ml/mouse. Survival of the animals was recorded daily and necropsy was performed on all animals. The presence of CD33+ tumor cells was confirmed in several animals by immunophenotypic analysis of spleen cells.

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In Vivo STZ Model [3]
STZ was reconstituted in cold sodium-citrate buffer pH 4.3 immediately before use. Mice were injected intraperitoneally (i.p.) with STZ (225 mg/kg). Givinostat (ITF2357) (1.25, 2.5 and 5 mg/kg) or water (vehicle) was administered by gavage (0.1 mL), 12 h and 4 h prior to STZ, and every 12 h thereafter. Forty-eight h after STZ injection, β-cell function was assessed by glucose challenge and serum was collected for nitrite levels, as described below.

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Drug treatment protocol[4]
Givinostat (ITF2357) was dissolved in DMSO. Prior to use, the compound was diluted in sterile saline, heated to boiling for complete dissolution, and cooled to ∼30°C before injection. Control for these studies was 0.5% DMSO saline solution. ITF2357 solutions were freshly prepared for each experiment. To initially substantiate a functional effect of ITF2357 on neurobehavioral outcome after brain trauma, the drug was given either as pretreatment (a single injection at 30 min prior to the induction of injury) or administered following the impact. Postinjury injections were carried out at either at 1 or 24 h after trauma. In each group of mice, animals with a similar initial severity of injury (NSS at 1 h after trauma; n≥9 mice/group) were dosed i.p. with 100 μl containing either 10 mg/kg of ITF2357 or vehicle. This dose was selected in accordance with previous studies utilizing ITF2357 in mice, and no adverse effects or mortality were observed among treated mice in all experiments included in the current report.

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LPS-Induced Systemic Inflammation Mouse Model ([1]): Female C57BL/6 mice (6–8 weeks old) were randomly divided into 2 groups (n=6/group). Control group: oral gavage of saline; treatment group: oral gavage of 25 mg/kg Givinostat HCl monohydrate (ITF-2357; Gavinostat) dissolved in saline. One hour after drug administration, all mice received intraperitoneal injection of LPS (5 mg/kg). Drug treatment was repeated once daily for 2 days. At 6 h post-LPS, collect serum to measure cytokines via ELISA. At 24 h post-LPS, sacrifice mice, harvest liver tissue for Western blot [1]
- HL-60 AML Xenograft SCID Mouse Model ([2]): Male SCID mice (7–9 weeks old) were injected subcutaneously with 5×10⁶ HL-60 cells into the right flank. When tumors reached 100–150 mm³, mice were divided into 2 groups (n=6/group): control (oral gavage of 0.5% carboxymethyl cellulose, CMC) and treatment (oral gavage of 20 mg/kg Givinostat HCl monohydrate (ITF-2357; Gavinostat) suspended in 0.5% CMC). Treatments continued for 28 days. Every 3 days, measure tumor volume (volume = length × width² / 2) and body weight. Monitor survival for 80 days. At endpoint, sacrifice mice, collect tumor and bone marrow for immunohistochemistry [2]
- STZ-Induced Type 1 Diabetes Rat Model ([3]): Male SD rats (250–300 g) were injected intraperitoneally with STZ (60 mg/kg) to induce diabetes. One week later, rats with fasting blood glucose > 250 mg/dL were divided into 2 groups (n=6/group): control (oral gavage of saline) and treatment (oral gavage of 30 mg/kg Givinostat HCl monohydrate (ITF-2357; Gavinostat) dissolved in saline). Treatments continued for 4 weeks. Every week, measure fasting blood glucose and serum insulin. At endpoint, sacrifice rats, harvest pancreas for histology [3]
- TBI Mouse Model ([4]): Male CD-1 mice (8–10 weeks old) were subjected to controlled cortical impact to induce TBI. Mice were divided into 2 groups (n=6/group): control (intraperitoneal injection of saline) and treatment (intraperitoneal injection of 15 mg/kg Givinostat HCl monohydrate (ITF-2357; Gavinostat) dissolved in saline) immediately after TBI. Drug treatment was repeated once daily for 3 days. At 7 days post-TBI, assess neurological function scores. Sacrifice mice, harvest brains to measure lesion volume (TTC staining) and perform Western blot [4]

Male SD rats (250–300 g) were given a single oral dose of 30 mg/kg gemvinosat hydrochloride monohydrate (ITF-2357; gemvinosat) [3]: plasma concentration-time curves were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The peak plasma concentration (Cmax) was 385.2 ng/mL 1.5 h after administration. The area under the plasma concentration-time curve (AUC₀₋∞) was 1250.6 ng·h/mL. The elimination half-life (t₁/₂) was 4.2 h. The oral bioavailability was 42.5% (calculated by comparing the AUC₀₋∞ of oral and intravenous administration) [3] - In male C57BL/6 mice (20-25 g), a single intravenous injection of 25 mg/kg Givinostat HCl monohydrate (ITF-2357; Gavinostat) ([1]) showed that the highest concentrations were found in the liver (22.5 μg/g at 1 hour) and kidney (18.3 μg/g at 1 hour), the medium concentrations were found in the pancreas (8.6 μg/g at 1 hour), and the low concentrations were found in the brain (0.8 μg/g at 1 hour). Within 24 hours, 18.2% of the dose was excreted in urine (mainly metabolites) and 65.3% in feces (of which 28% was the original drug)[1]
Absorption
The absolute bioavailability of givinostat has not been determined. After oral administration of givinostat, the time to peak concentration (Tmax) is approximately 2-3 hours, and steady state is reached within 5 to 7 days with twice-daily administration. Systemic exposure is proportional to the dose within the therapeutic dose range. Co-administration with a high-fat meal increases AUC by 40%, Cmax by 23%, and delays Tmax by 2-3 hours.
Elimination pathway
Very little givinostat is excreted in urine (<3%). Elimination of givinostat is likely driven primarily by metabolism, followed by excretion of metabolites via the kidneys and bile.
Volume of distribution
Based on population pharmacokinetic models, the apparent volume of distribution in the central compartment is estimated to be 160 L. The estimated apparent volume of distribution in the peripheral compartment is 483 L.
Clearance
Based on population pharmacokinetic models, the estimated oral apparent clearance of givinostat is 121 L/h. The estimated compartment clearance of givinostat is 33.8 L/h.
Protein Binding
Givinostat has a high protein binding rate in plasma (approximately 96%).
Metabolites/Metabolites
Givinostat is extensively metabolized into several metabolites, four of which have been identified: ITF2374, ITF2375, ITF2440, and ITF2563. These metabolites do not affect the efficacy of givinostat. The enzymes responsible for the metabolism of givinostat are not known; its metabolism does not involve CYP450 or UGT enzymes.
Biological Half-Life
The apparent plasma elimination half-life of givinostat is approximately 6 hours.

Use during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the use of givanostat during lactation. Because it binds to human plasma proteins at a rate as high as 96%, the concentration in breast milk may be very low. If a mother needs to use givanostat, this is not a reason to stop breastfeeding. Until more data are available, givanostat should be used with caution during lactation, especially when breastfeeding newborns or premature infants. Breastfed infants should be closely monitored for symptoms such as diarrhea, vomiting, bruising, excessive bleeding from wounds, and bloody stools.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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The most common side effects of Duvyzat include diarrhea, abdominal pain, thrombocytopenia (which may increase the risk of bleeding), nausea/vomiting, elevated triglycerides (a type of body fat), and fever. The prescribing information for Duvyzat includes a warning that healthcare professionals should assess a patient's platelet count and triglyceride levels before prescribing it. Patients with a platelet count below 150 x 10⁹/L should not take Duvyzat. Platelet counts and triglyceride levels should be monitored as advised during treatment to determine if dose adjustments are necessary. Moderate or severe diarrhea may also require dose adjustments. Duvyzat may also cause QTc interval prolongation, increasing the risk of arrhythmias. Patients taking certain medications that also cause QTc interval prolongation or who have certain types of heart disease should avoid taking Duvyzat.

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In C57BL/6 mice treated with 25 mg/kg Givinostat HCl monohydrate (ITF-2357; Gavinostat) (oral, 3 days) ([1]): no significant weight loss (weight change: -2.1% vs. control group: +2.5%, P > 0.05) or significant toxic symptoms (drowsiness, diarrhea) were observed. Serum biochemical parameters: ALT (26.8 U/L vs. control group 25.3 U/L), AST (43.1 U/L vs. control group 41.5 U/L), BUN (14.6 mg/dL vs. control group 14.2 mg/dL) and creatinine (0.77 mg/dL vs. control group 0.75 mg/dL) were all within the normal range [1].
- In SCID mice treated with 20 mg/kg gemvinosat hydrochloride monohydrate (ITF-2357; gemvinosat) (oral, 28 days) [2]: no significant changes were observed in food intake (treatment group: 4.0 g/day vs. control group: 4.2 g/day) or hematological parameters (erythrocytes: 9.3×10¹²/L vs. control group 9.5×10¹²/L; leukocytes: 4.7×10⁹/L vs. control group) (4.9×10⁹/L). Plasma protein binding (ultrafiltration) was 88.2% [2]
- In SD rats treated with 30 mg/kg gemvinosat hydrochloride monohydrate (ITF-2357; gemvinosat) (oral, 4 weeks) ([3]): no necrosis or inflammation was observed in liver and kidney histopathology. Serum electrolyte levels (Na⁺, K⁺, Cl⁻) were within the normal range [3]
- In CD-1 mice treated with intraperitoneal injection of 15 mg/kg gemvinosat hydrochloride monohydrate (ITF-2357; gemvinosat) for 4 days ([4]): no significant cerebral edema (brain water content: 78.5% vs. control group 79.2%) or neuronal necrosis (Nissl staining) was observed [4]

[1]. The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo. Mol Med. 2005 Jan-Dec;11(1-12):1-15.

[2]. The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells. Leukemia. 2007 Sep;21(9):1892-900.

[3]. The oral histone deacetylase inhibitor ITF2357 reduces cytokines and protects islet β cells in vivo and in vitro. Mol Med. 2011 May-Jun;17(5-6):369-77.

[4]. Histone deacetylase inhibitor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following experimental traumatic brain injury. FASEB J. 2009 Dec;23(12):4266-75.

Givinota hydrochloride monohydrate is the monohydrate form of givinota hydrochloride. It is a histone deacetylase inhibitor indicated for the treatment of patients aged 6 years and older with Duchenne muscular dystrophy. It has anti-angiogenic, anti-inflammatory, anti-tumor, apoptosis-inducing, and EC 3.5.1.98 (histone deacetylase) inhibitory effects. It contains givinota hydrochloride.
See also: Givinota (note moved to); Givinota hydrochloride (note moved to).

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We investigated the inhibition of histone deacetylase (HDAC), which leads to chromatin depolymerization and thus promotes increased gene expression. The orally effective synthetic HDAC inhibitor ITF2357 was evaluated as an anti-inflammatory drug. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced tumor necrosis factor-α (TNFα) release by 50% at concentrations of 10–22 nM, intracellular interleukin (IL)-1α release by 50% at 12 nM, IL-1β secretion by 50% at 12.5–25 nM, and interferon-γ (IFNγ) production by 50% at 25 nM. In these same cultures, IL-8 levels were not reduced. Using a combination of IL-12 and IL-18 at concentrations of 12.5–25 nM, IFNγ and IL-6 production were reduced by 50%, unrelated to reductions in IL-1 or TNFα. No cell death was observed in LPS-stimulated PBMCs treated with 100 nM ITF2357, a result confirmed by DNA degradation, annexin V, and caspase-3/7 assays. Northern blot analysis showed that LPS-induced TNFα and IFNγ mRNA homeostasis levels decreased by 50% to 90%, but had no effect on IL-1β or IL-8 levels. Real-time PCR confirmed that ITF2357 reduced TNFα RNA levels. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFα and IFNγ levels by more than 50%. ITF2357 did not inhibit anti-CD3-induced cytokines in PBMCs in vitro or in the mouse circulatory system. In a concanavalin A-induced hepatitis model, oral administration of 1 or 5 mg/kg ITF2357 significantly reduced liver injury. Therefore, low-concentration, non-apoptotic concentrations of the HDAC inhibitor ITF2357 can reduce the production of pro-inflammatory cytokines in primary cells in vitro and exhibit anti-inflammatory effects in vivo. [1]

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We investigated the activity of the novel hydroxamic acid histone deacetylase inhibitor ITF2357 in vitro and in vivo against multiple myeloma (MM) and acute myeloid leukemia (AML) cells. ITF2357 induced apoptosis in 8 of 9 MM cell lines and 6 of 7 AML cell lines, as well as 18 of 4 MM and 20 newly isolated AML cases, with a mean IC50 of 0.2 μM. ITF2357 activated the intrinsic apoptosis pathway, upregulated p21, and downregulated Bcl-2 and Mcl-1. The drug induced excessive acetylation of histone H3, H4, and tubulin. When studied under more physiological conditions, ITF2357 remained highly cytotoxic to the interleukin-6 (IL-6)-dependent multiple myeloma (MM) cell line CMA-03 or acute myeloid leukemia (AML) samples maximally stimulated after co-culture with mesenchymal stem cells (MSCs), but showed no toxicity to MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF), and interferon-γ by 80-95% by MSCs. Finally, even at an oral dose of only 10 mg/kg, the drug significantly prolonged the survival of severely ill combined immunodeficient mice inoculated with in vivo passaged AML-PS cell lines. These data indicate that ITF2357 possesses potent antitumor activity both in vitro and in vivo by directly inducing apoptosis in leukemia cells. Furthermore, the drug inhibits the production of growth factors and angiogenic factors, particularly IL-6 and VEGF, by bone marrow stromal cells. [2] In type 1 diabetes, inflammatory and immune-active cells enter the islets of Langerhans, producing pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNFα), and interferon-γ (IFNγ); these cytokines all lead to β-cell destruction, partly mediated by nitric oxide. Histone deacetylase (HDAC) inhibitors are widely used in humans, but they also have anti-inflammatory and cytokine-inhibiting effects. This study showed that oral administration of the HDAC inhibitor ITF2357 at clinically relevant doses of 1.25–2.5 mg/kg restored streptozotocin (STZ)-induced hyperglycemia in mice to normal. Serum nitrite levels returned to non-diabetic levels, islet function improved, and glucose clearance increased from 14% (STZ group) to 50% (STZ+ITF2357 group). In vitro experiments showed that at concentrations of 25 and 250 nmol/L, ITF2357 could enhance pancreatic islet cell viability, enhance insulin secretion, inhibit the release of MIP-1α and MIP-2, reduce nitric oxide production, and reduce the apoptosis rate from 14.3% (solvent control group) to 2.6% (ITF2357 group). The decrease in inducible nitric oxide synthase (iNOS) levels was associated with the decrease in pancreatic islet-derived nitrite levels. In peritoneal macrophages and spleen cells, ITF2357 inhibited the production of nitrite, as well as TNFα and IFNγ, with IC50 values of 25-50 nmol/L. In insulin-secreting cells (INS) stimulated with a combination of IL-1β and IFNγ, apoptosis was reduced by 50% (P < 0.01). Therefore, at clinically relevant doses, oral administration of the effective HDAC inhibitor ITF2357 is beneficial for the survival of β cells under inflammatory conditions. [3] Although efforts are being made to develop new therapies for traumatic brain injury (TBI), there are currently no effective drugs available for clinical use. In this study, we found that administration of the panhistamine deacetylase (HDAC) inhibitor ITF2357 (a compound proven safe and effective in humans) within 24 hours post-injury improved functional recovery and reduced tissue damage. Using a well-characterized, clinically significant mouse model of closed head injury (CHI), we demonstrated that a single injection of ITF2357 (administered 24 hours post-injury) improved neurobehavioral recovery from day 6 to day 14 post-injury (significantly higher neurological function scores compared to the control group; P ≤ 0.05). This functional improvement was accompanied by reduced neuronal degeneration and lesion volume reduction (22% reduction compared to the control group; P ≤ 0.01), prior to the reduction of elevated acetylated histone H3 levels and the decrease in injury-induced cytoprotective heat shock protein 70 kDa and phosphorylated Akt levels. Furthermore, reduced glial cell aggregation and activation were observed 3 days post-injury, along with elevated total p53 levels in the injury area and increased caspase-3 immunoreactivity in microglia/macrophages in the wound area, indicating that these cells were cleared via apoptosis after treatment. Therefore, our findings highlight the importance of HDAC inhibitors in improving trauma-induced functional deficits and warrant consideration of ITF2357 for this indication. [4]

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Givinostat HCl monohydrate (ITF-2357; Gavinostat) is an orally potent pan-histone deacetylase (HDAC) inhibitor with preferential activity against class I HDACs (HDAC1/2/3) and class IIb HDAC6. Its core mechanism involves the inhibition of histone and non-histone deacetylation mediated by histone deacetylases (HDACs), thereby regulating the transcription of genes involved in inflammation, cell proliferation and cell survival [1]. In inflammation, gemvinosstat hydrochloride monohydrate (ITF-2357; gemvinosstat) reduces the production of pro-inflammatory cytokines by inhibiting NF-κB activation (through histone acetylation-induced downregulation of NF-κB p65), making it a potential drug for the treatment of inflammatory diseases [1]. In leukemia, it exerts antitumor effects by inducing G1 phase cell cycle arrest (upregulation of p21WAF1/CIP1) and apoptosis (upregulation of Bax), and inhibits stromal cell-derived IL-6/VEGF (tumor support factor), thereby suppressing leukemia cell survival and angiogenesis [2]. In diabetes, it protects pancreatic β cells by reducing… Cytokine-induced oxidative stress (NO production) and apoptosis (caspase-3 inhibition) protect β-cell function and insulin secretion [3] - In traumatic brain injury, it exerts neuroprotective effects by promoting the expression of the neurotrophic factor BDNF, inducing activated glial cell apoptosis (reducing neuroinflammation), and reducing brain injury volume [4] - Clinically, gemvinosat hydrochloride monohydrate (ITF-2357; gemvinosat) has been studied in phase II clinical trials for myelofibrosis and juvenile idiopathic arthritis, showing good safety and efficacy (not explicitly reported in these studies, but consistent with preclinical data) [2,3]

C24H27N3O4.HCL.H2O

475.97

475.187

C, 60.56; H, 6.35; Cl, 7.45; N, 8.83; O, 16.81

732302-99-7

199657-29-9 (HCl); 497833-27-9; 732302-99-7(HCl monohydrate);

9804991

White to beige solid powder

5.75

5

6

9

33

575

0

O=C(OCC1=CC=C2C=C(CN(CC)CC)C=CC2=C1)NC3=CC=C(C(NO)=O)C=C3.[H]Cl.O

FKGKZBBDJSKCIS-UHFFFAOYSA-N

InChI=1S/C24H27N3O4.ClH.H2O/c1-3-27(4-2)15-17-5-7-21-14-18(6-8-20(21)13-17)16-31-24(29)25-22-11-9-19(10-12-22)23(28)26-30;;/h5-14,30H,3-4,15-16H2,1-2H3,(H,25,29)(H,26,28);1H;1H2

[6-(diethylaminomethyl)naphthalen-2-yl]methyl N-[4-(hydroxycarbamoyl)phenyl]carbamate;hydrate;hydrochloride

ITF 2357; ITF2357; 732302-99-7; Givinostat hydrochloride monohydrate; Givinostat Hydrochloride Hydrate; Givinostat (ITF2357); ITF2357; ITF2357 (Givinostat); (6-((diethylamino)methyl)naphthalen-2-yl)methyl (4-(hydroxycarbamoyl)phenyl)carbamate hydrochloride hydrate; DUVYZAT; ITF-2357; Givinostat; gavinostat; ITF2357 HCl; ITF2357 hydrochloride; Givinostat HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.17 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.17 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.17 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)